The present invention relates, in general, to materials useful as components of cytotoxic therapeutic agents. More particularly, the invention relates to analogs of ribosome-inactivating proteins specifically modified for conjugation to targeting molecules and to polynucleotides encoding ribosome-inactivating proteins and analogs thereof.
Ribosome-inactivating proteins (RIPs) comprise a class of proteins which is ubiquitous in higher plants. RIPs have also been isolated from bacteria. RIPs are potent inhibitors of eukaryotic protein synthesis. The N-glycosidic bond of a specific adenine base is hydrolytically cleaved by RIPs in a highly conserved loop region of the 28S rRNA of eukaryotic ribosomes thereby inactivating translation.
Stirpe et al., FEBS Lett., 195(1,2), 1-8 (1986) groups plant RIPs into two types. Type I proteins each consist of a single peptide chain having ribosome-inactivating activity, while Type II proteins each consist of an A-chain, essentially equivalent to a Type I protein, disulfide-linked to a B-chain having cell-binding properties. Gelonin, dodecandrin, tricosanthin, tricokirin, bryodin, Mirabilis antiviral protein (MAP), barley ribosome-inactivating protein (BRIP), pokeweed antiviral proteins (PAPs), saporins, luffins and momordins are examples of Type I RIPs, while ricin and abrin are examples of Type II RIPs. Amino acid sequence information is reported for various ribosome-inactivating proteins. It appears that at least the tertiary structure of active sites is conserved among Type I RIPs, bacterial RIPs and A-chains of Type II RIPs and, in many cases, primary structure homology is also found. Ready et al., J. Biol. Chem., 259(24), 15252-15256 (1984) and other reports suggest that the two types of RIPs are evolutionarily related.
Separated from their natural environment, Type I plant ribosome-inactivating proteins may be particularly suited for use as components of cytotoxic therapeutic agents. A RIP may be conjugated to a targeting agent that will deliver the RIP to a particular cell type in vivo in order to selectively kill those cells. Because some RIPs, such as the Type I RIP gelonin, are only available from scarce plant materials, it is desirable to clone the genes encoding the RIPs to enable recombinant production of the proteins. It is also desirable to develop analogs of the natural proteins which may be easily conjugated to targeting molecules while retaining their natural biological activity because most Type I RIPs have no natural sites (i.e. available cysteine residues) for conjugation to targeting agents.
There thus exists a need in the art for cloned genes encoding ribosome-inactivating proteins and for analogs of ribosome-inactivating proteins which may be easily conjugated to targeting molecules.